Abstract:

Mycobacterium tuberculosis (M. tb) has been shown to be the main causative agent of
tuberculosis in elephants worldwide. M. tb may be transmitted from infected humans to other
species including elephants and vice versa, in case of prolonged intensive contact. An
accurate diagnostic approach covering all phases of the infection in elephants is required. As
M. tb is an intracellular pathogen and cell-mediated immune (CMI) responses are elicited
early after infection, the skin test is the CMI assay of choice in humans and cattle. However,
this test is not applicable in elephants. The interferon gamma (IFN-γ) assay is considered a
good alternative for the skin test in general, validated for use in cattle and humans. This study
was aimed at development of an IFN-γ assay applicable for diagnosis of tuberculosis in
elephants. Recombinant elephant IFN-γ (rEpIFN-γ) produced in eukaryotic cells was used to
immunize mice and generate the monoclonal antibodies. Hybridomas were screened for IFN-
γ-specific monoclonal antibody production and subcloned, and antibodies were isotyped and
affinity purified. Western blot confirmed recognition of the rEpIFN-γ. The optimal
combination of capture and detection antibodies selected was able to detect rEpIFN-γ in
concentrations as low as 1 pg/ml. The assay was shown to be able to detect the native
elephant IFN-γ, elicited in positive-control cultures (pokeweed mitogen (PWM), phorbol
myristate acetate plus ionomycin (PMA/I)) of both Asian and African elephant whole-blood
cultures (WBC). Preliminary data were generated using WBC from non-infected elephants, a
M. tb infection-suspected elephant and a culture-confirmed M. tb-infected elephant. The latter
showed measurable production of IFN-γ after stimulation with ESAT6/CFP10 PPDB and
PPDA in concentration ranges as elicited in WBC by Mycobacterium tuberculosis complex
(MTBC)-specific antigens in other species. Hence, the IFN-γ assay presented potential as a diagnostic tool for the detection of elephant tuberculosis. Validation of the assay will require
its application in large populations of non-infected and infected elephants.